Members of the ErbB receptor tyrosine kinase family such as EGF receptor and ErbB2 play critical roles in uncontrolled cell proliferation and invasiveness, hallmarks of oncogenic transformation in breast and other epithelial cancers. We and others have identified Cbl family of ubiquitin ligases as negative regulators of EGF receptor and other tyrosine kinases involved in EGF receptor signaling. Our recent work has identified a novel role of Cbl proteins in regulating the VAV guanine nucleotide exchange factors (GEFs) that link EGFR to cell proliferation, migration and tumor invasiveness through Rho/Rac small GTPases. Based on our initial studies, we hypothesize that Cbl family ubiquitin ligases represent crucial regulators of EGF receptor-mediated VAV signaling and provide a barrier to oncogenic transformation of human mammary epithelial cells. Here, we propose comprehensive strategies to test these hypotheses using a human mammary epithelial cell system with direct relevance to understanding breast cancer etiology. We will use genetic knockdown, overexpression and dominant-negative mutant expression to identify critical EGF receptor-activated biological responses of human mammary epithelial cells that are critically dependent on VAV GEFs. We will then alter the expression and/or function of Cbl proteins to test the notion that Cbl proteins are direct negative regulators of VAV GEFs. We will carry cell biological, molecular and biochemical analyses to assess the mechanisms by which Cbl-mediated ubiquitin modification controls the fate of activated VAV GEFs. Finally, we will use in vitro and in vivo analyses to test the hypothesis that Cbl-mediated negative regulation of VAV GEFs provides a barrier for oncogenic transformation of human mammary epithelial cells into breast cancer cells. Through this comprehensive approach, we hope to elucidate the biological significance and oncogenesis-related roles of a novel tyrosine kinase regulatory pathway that is based on ubiquitinylation and degradation of critical components of EGF receptor signaling machinery. Success of these studies is likely to open new therapeutic avenues for EGF receptor- and other tyrosine kinase-driven malignancies such as breast cancer.